CN112469447A

CN112469447A - Intranasal delivery of fluorescent markers

Info

Publication number: CN112469447A
Application number: CN201980027922.2A
Authority: CN
Inventors: 本杰明·迈克尔·戴维斯; 玛丽亚·弗朗西斯卡·科迪里奥; 郭丽
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2018-04-23
Filing date: 2019-04-23
Publication date: 2021-03-09
Also published as: SG11202010320RA; CA3097661A1; JP2021522215A; WO2019207287A1; US20210228745A1; EP3784293A1; AU2019259198A1; GB201806581D0

Abstract

The invention provides a method of diagnosing a CNS disorder comprising administering to a subject a fluorescent marker of retinal integrity and generating an image of the eye of the subject, wherein delivery of the fluorescent marker by intranasal administration is also provided, as well as intranasal administration of a fluorescent marker of retinal integrity for use in the method. Furthermore, the present invention provides a pharmaceutical composition comprising annexin or a functional fragment or derivative thereof bound to a compound of 2kDa or less, wherein the pharmaceutical composition comprises annexin or a functional fragment or derivative thereof bound at a concentration of at least mg/ml.

Description

Intranasal delivery of fluorescent markers

Technical Field

The present invention relates to fluorescent markers for the diagnosis of CNS diseases.

Background

Retinal assessments can provide information about the presence and severity of many systemic diseases. For example, Detection of Apoptotic Retinal Cells (DARC) describes a technique to monitor the rate of retinal cell death, and phase I clinical trials to diagnose glaucoma have recently been completed (coledlor et al, 2017), and a series of phase II clinical trials are currently in progress for neurodegenerative diseases, including glaucoma, age-related macular degeneration, optic neuritis, and down's disease (as models of alzheimer's disease). This technology currently involves the intravenous administration of a novel fluorescer called Anx776, which comprises a modified version of the endogenous protein Annexin a5, which is fluorescently conjugated with the near infrared fluorophore Dy-776 (keldello et al, 2017).

DARC takes advantage of the unique optical properties of the eye and can directly observe single nerve cell apoptosis in patients using fluorescently labeled human annexin V derivatives. Annexin V is a human protein that has the ability to bind to Phosphatidylserine (PS) in the presence of calcium. PS is present in the plasma membrane of each cell, however, apoptotic cells express PS in the outer leaflet of the plasma membrane. Annexin V binds to the exposed PS, recognizing apoptosis.

For both phase I and phase II clinical trials of DARC, Anx776 was administered intravenously. Other fluorescent molecules used to detect retinal disease by confocal scanning laser ophthalmoscopy (cSLO) imaging are also administered primarily by intravenous injection. For example, fluorescein sodium and indocyanine green (ICG) are commonly administered intravenously to label retinal vasculature to identify abnormal vascular growth (angiogenesis) and leakage associated with age-related macular degeneration (AMD) (jozike et al, 2005).

The use of intravenous administration requires direct medical supervision, meaning that the patient is not routinely evaluated unless a suspected pathology is suspected. Therefore, there is a need for alternative techniques that can be used to assess the retina, to enable patients to make such assessments more extensive and possibly to diagnose retinal and neurodegenerative diseases earlier.

Disclosure of Invention

According to a first aspect of the invention, there is provided a fluorescent marker of retinal integrity for use in diagnosing a Central Nervous System (CNS) disease, wherein the fluorescent marker is delivered by intranasal administration. Surprisingly, the inventors of the present application found that intranasal administration of a fluorescent label results in a rapid accumulation of fluorescence in the retina, with the results of retinal imaging being the same as those obtained after a previous intravenous administration of the same fluorescent agent.

Without being bound by theory, the inventors believe that intranasally delivered fluorescent markers of retinal integrity are systemically absorbed into the circulation. Due to local administration, fluorescent labeling of retinal integrity may require reduced dosages and may be more rapid in onset than systemic administration of the same agent.

As used herein, a fluorescent marker of retinal integrity refers to a fluorescent marker used to interrogate the health of retinal cells and/or the integrity of retinal cells and/or blood vessels. For example, fluorescent labels may identify and/or distinguish between apoptotic and necrotic cells, or identify regions of vascular leakage or angiogenesis. In response to excitation, the fluorescent label emits light and may have an emission wavelength of about 400nm to about 1000nm, preferably about 500nm to about 900 nm.

The fluorescent marker of retinal integrity is preferably provided in a form suitable for local delivery, particularly intranasal delivery. The fluorescent marker of retinal integrity may be provided in the form of a pharmaceutical composition, in the form of a solution, suspension or dry powder suitable for inhalation. The fluorescently labeled pharmaceutical composition comprising retinal integrity can be sterile and can comprise one or more pharmaceutically acceptable carriers or excipients. Suitable carriers and excipients will be familiar to those skilled in the art and may be optimized according to the intended route of intranasal delivery. For example, a composition comprising a fluorescent marker of retinal integrity may further comprise a buffer, a binder, a preservative, a thickening agent, or an antioxidant, such as trehalose.

In some embodiments of the invention, the fluorescent marker of retinal integrity may be a fluorescent marker of retinal vascular integrity. Typically, the fluorescent label will enter and circulate within retinal blood vessels, thereby facilitating observation of abnormal sites of blood vessel growth (angiogenesis) and/or leakage.

The fluorescent marker of retinal vascular integrity may have a molecular weight of about 2kDa or less, or 1kDa or less. In some embodiments of the invention, the fluorescent marker of retinal vascular integrity may have a molecular weight of about 100Da, or about 1kDa, preferably a molecular weight of about 300Da to about 800 Da.

Suitable fluorescent labels for retinal vascular integrity for use in the present invention include fluorophores, with particularly preferred fluorescent labels for retinal vascular integrity including sodium fluorescein and indocyanine green (ICG).

As noted above, the fluorescent marker of retinal vascular integrity will be administered intranasally, and may be administered at the same or less dose as the same marker administered intravenously. For example, fluorescein sodium can be administered intranasally at a concentration of about 50mg/mL to about 500mg/mL, preferably at a concentration of about 50mg/mL to about 200 mg/mL. In some embodiments of the invention, fluorescein sodium can be administered intranasally at a concentration of about 100 mg/mL. ICG may be administered intranasally at a concentration of from about 1mg/mL to about 100mg/mL, preferably from about 25mg/mL to about 100 mg/mL. In some embodiments of the invention, the ICG may be administered intranasally at about 50 mg/mL.

In some embodiments of the invention, the fluorescent marker of retinal integrity may be a marker of retinal cell integrity. Typically, fluorescent markers of retinal cell integrity include fluorescent tags and one or more markers of apoptosis, necrosis, cellular activity, cellular stress, or protein aggregation.

Fluorescent labels refer to compounds or molecules (e.g., fluorophores) that emit light in response to excitation, which may be selected for use because of increased signal-to-noise ratio, thereby increasing image resolution and sensitivity while complying with exposure safety standards, avoiding phototoxic effects. Preferably, the fluorescent label causes little or no inflammation when administered. The fluorescent label may have a wavelength in the infrared or near infrared range. The fluorescent tag may have an emission wavelength of about 400nm to about 1000nm, preferably about 500nm to about 900nm, more preferably about 700nm to about 900 nm.

Suitable fluorescent labels include one or more of fluorescein sodium, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488, and D-781. In a preferred embodiment of the present invention, the fluorescent tag is Dy-776.

Fluorescent retinal cell integrity markers can be prepared using standard techniques for combining fluorescent labels with markers. Such labels are available from well known sources, such as Dyomics. Suitable techniques for combining the label with the tag are known in the art and may be provided by the manufacturer of the label.

An apoptosis marker refers to a marker that distinguishes cells undergoing apoptosis from living cells. Furthermore, the marker should preferably be able to distinguish apoptotic cells from necrotic cells. For example, it may be a compound or molecule that specifically binds apoptotic cells but not living or necrotic cells. For example, apoptosis markers include annexin family proteins. Annexins are proteins that bind reversibly to the cell membrane in the presence of cations. In particular, annexin is capable of binding Phosphatidylserine (PS) in the presence of calcium. PS is present in the plasma membrane of each cell, however, apoptotic cells express PS in the outer leaflet of the plasma membrane. Annexin binds to exposed PS, thereby recognizing apoptosis. The annexin useful in the invention may be natural or recombinant. The protein may be intact or functional fragments, that is, fragments or portions of annexin specifically bind to the same molecule as the entire protein. Furthermore, functional derivatives of such proteins may be used. In particular, functional fragments or derivatives of annexin may include molecules containing an "annexin repeat," a domain of about 70 amino acids that is conserved both within a single annexin and between family members. A variety of annexins are available, such as the annexin described in U.S. patent application publication No. 2006/0134001 a. The preferred annexin is annexin 5, which is well known in the art. Other annexins that may be used include

annexins

11, 2 and 6. Other apoptotic markers are known in the art, including, for example, the C2A domain of synaptotagmin I, duramycin, non-peptide based isatin sulfonamide analogs such as WC-II-89 and ApoSense, such as NST-732, DDC, and ML-10 (saint hubert et al, 2009).

In a preferred embodiment of the invention, the marker of apoptosis is annexin 128 (teter et al, 2005). Annexin 128 is a variant of annexin 5 that differs from the wild type by two single amino acid mutations. Annexin 128 includes an exposed thiol group at the N-terminus, thereby increasing the binding efficiency of the molecular tag (e.g., fluorescent label).

In a preferred embodiment of the present invention, the fluorescent marker for retinal cell integrity comprises annexin 128 bound to Dy-776, and the annexin 128 and Dy-776 may be bound at a fluorescent Labach marker ratio of 1: 1.

Necrosis markers are markers that distinguish cells undergoing necrosis from living cells, cells undergoing apoptosis. For example, it may be a compound or molecule that specifically binds to necrotic cells but not to live or apoptotic cells. Markers of necrosis include Propidium Iodide (PI), an intercalating agent that binds nucleic acids with little or no sequence preference. Other necrosis markers are known in the art and include pyrophosphate, antimyosin, gluconate, hypericin and its derivatives, such as hypericin monocarboxylic acid and palmitic acid, e.g., bis-hydrazide-bis DTPA palmitic acid. In particular, 99 mTc-pyrophosphate, 111-antimyosin, 99 mTc-gluconate, and methylene blue were used.

Other indications of cellular activity may also be used to identify apoptotic or necrotic cells. For example, a change in mitochondrial function can be observed, Reactive Oxygen Species (ROS) can be used as a marker, and calcium ions can be used as a marker. Markers of cellular activity may include one or more of membrane dyes, mitochondrial dyes, autophagy dyes, necrosis dyes, and calcium flux. More particularly, the marker of cellular activity may comprise one or more of Fluo-3, N- (fluorescein-5-thiocarbamoyl) -1, 2-hexacosanoyl-sn-glycero-3-phosphoethanolamine, JC-1, dual emission JC-9, reduced rhodamine and romimine, rhodamine 123 and Di-8-ANePS. Markers of cellular stress may include one or more markers of lipid peroxidation, Glutathione (GSH), or Reactive Oxygen Species (ROS), such as superoxide, peroxyl radicals, hydrogen peroxide, hydroxyl, and peroxynitrite.

Protein aggregation in the retina can occur within or outside cells within or around retinal neurons, but typically occurs outside of the retinal vasculature. The presence of protein aggregates is known to be associated with cells undergoing neurodegeneration. Suitable protein aggregation markers include one or more of congo red, curcumin or thioflavin S.

As noted above, the present invention provides intranasally delivered fluorescent markers of retinal integrity for use in diagnosing CNS disorders in a subject. The subject is preferably a mammal, including a human, and may be a pediatric or geriatric patient. CNS disorders can be identified by analysis of retinal integrity, see methods described in WO2009/077750 and WO 2011/055121. For example, fluorescent labeling of retinal integrity can be used to show the distribution of cell death in the retina by labeling apoptotic and/or necrotic cells, from which different neurodegenerative diseases with different patterns of apoptotic and/or necrotic activity can be distinguished.

The central nervous system disorder may be inflammatory (e.g., arthritis or granuloma), infectious (e.g., virus, encephalitis, or bacteria), vascular (e.g., angiogenesis, occlusion, or metabolism), or degenerative (e.g., glaucoma, age-related macular degeneration (AMD), Alzheimer's disease, or Parkinson's disease). In some embodiments of the invention, the CNS disorder can be a neurodegenerative disease, in particular an ocular neurodegenerative disease. The term "ocular neurodegenerative disease" is well known to those skilled in the art and refers to diseases caused by gradual and progressive loss of ocular neurons, including but not limited to glaucoma, AMD, optic neuritis, and diabetic retinopathy. Neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, and Friedrich's ataxia. In some embodiments of the invention, CNS disorders can include craniocerebral trauma, stroke, cerebral palsy (e.g., cerebral palsy caused by neonatal hypoxia), and cancer (including brain tumors).

The invention also provides a method for diagnosing a CNS disease comprising administering to a patient a fluorescent marker of retinal integrity as described herein and generating an image of the retina of the patient, wherein the fluorescent marker of retinal integrity is administered intranasally. Images of the patient's retina are preferably generated in vivo and may be obtained using cSLO imaging. The presence or absence of a fluorescent signal in the image may indicate the presence or absence of a CNS disease, thereby enabling a clinician to diagnose the presence or absence of a disease. If a fluorescent signal is present in the image, the location and distribution of the fluorescence can allow the clinician to distinguish the type of central nervous system disease. For example, the fluorescent signal may be indicative of a site of angiogenesis or vascular leakage. Alternatively, the fluorescent signal may be indicative of the distribution pattern and/or number of apoptotic and/or necrotic cells.

The diagnostic methods of the invention may also be used to monitor the progression of a disease, for example, to assess the effectiveness of a treatment or stage a disease. A first in vivo image of a patient's eye may be compared to a subsequent in vivo image of the same patient's eye, the subsequent in vivo image being taken days, weeks or months after the first image. Changes in the fluorescent signal can indicate disease progression or an effective treatment. For example, an increase in fluorescence signal may indicate an increase in the number of apoptotic cells, possibly indicating disease progression. The images can be analysed as described in WO 2011/055121.

In another aspect of the invention, the invention provides a pharmaceutical composition comprising a marker of retinal cell integrity as described above, wherein the pharmaceutical composition comprises annexin or a functional fragment or derivative thereof bound to a compound of 2kDa or less, wherein the annexin or the functional fragment or derivative thereof is present at a concentration of at least 5 mg/ml. Compositions comprising annexin suffer from precipitation problems when the annexin is present at a concentration of about 2mg/ml or higher. However, the inventors of the present application have been able to solve this problem in order to provide a composition comprising annexin at a concentration of at least 5 mg/ml. The composition has proven to be stable to storage at 25 ℃ for up to 50 days in the absence of light. Without being bound by theory, the inventors believe that binding of a compound of 2kDa or less to the N-terminus of annexin may sterically hinder protein aggregation.

The binding may be electrostatic or covalent, the compound preferably being bound to the N-terminus (i.e. the first 30 amino acids) of annexin or a functional fragment or derivative thereof. In some embodiments of the invention, the annexin or the functional fragment or derivative thereof may be present in a concentration of about 5mg/ml to about 20mg/ml, preferably about 5mg/ml to about 10 mg/ml.

The compound may be an organic compound or an inorganic compound, and may have a size of about 100Da to about 2kDa, preferably about 200Da to about 1kDa, more preferably about 300Da to about 800 Da. The compound may be a fluorescent label as described above, or may be a therapeutic agent. Suitable fluorescent labels may be selected from one or more of sodium fluorescein, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488, and D-781. In a preferred embodiment of the present invention, the fluorescent label is Dy-776.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises annexin 128 bound to Dy 776. Surprisingly, several methods of concentrating markers of retinal cell integrity have been evaluated, and the results demonstrate that annexin 128 bound to Dy776 is more stable at high concentrations than annexin 5.

The pharmaceutical composition is preferably suitable for intranasal administration (e.g. as a solution, suspension or dry powder suitable for inhalation) and may comprise suitable carriers and/or excipients as described above. The pharmaceutical composition may be in the form of a lyophilized powder and may be administered to the patient as a dry powder suitable for inhalation. Alternatively, the lyophilized powder can be rehydrated to form a suspension prior to administration.

Drawings

The invention will now be described in detail, by way of example only, with reference to the accompanying drawings.

Figure 1 intranasal administration of Anx776 rapidly reached the mouse retina and apoptotic retinal cells were labeled. cSLO images of the same C57BL/6J mouse eyes 3 hours after baseline [ A, B ], and intranasal administration of Anx776[ C, D ] and intravitreal 4% DMSO [ B, D ]. Note that the appearance of apoptosis (white spots) was seen in the eye only in the case of intravitreal injection of 4% DMSO [ D ]. [E] DARC points were trimmed 3h after baseline and IN Anx776 dosing. [F] Phosphatidylserine binding assays showed that freeze-dried concentrated Anx776 maintained good PS binding activity after 50 days of storage at 25 ℃ while being protected from light (EC50 ratio <2) compared to fresh Anx776 material.

FIG. 2 cSLO images after administration of 0.025mL sodium fluorescein (20% w/v) in C57BL/6J mice (488nm challenge).

FIG. 3 intranasal administration of 0.025mL indocyanine green (ICG) (795nm challenge) in C57BL/6J mice.

Examples

Example a) rapid arrival in circulation and retina following intranasal administration of Anx776

Annexin 128 binds to Dy-776 in its maleimide form, providing a marker for retinal cell integrity, designated Anx 776. The Anx776 preparation currently used in clinic (0.2mg/mL) was concentrated 25-fold to 5mg/mL using a 5kDa MWCO filter in a buffer containing 20mM sodium citrate, 280mM glucose, pH 6.2 water for injection.

Concentrated Anx776(hiAnx776) was tested in vivo. Using the established model [6], intravitreal injection of 4% DMSO (1pL injection, PBS buffer) was used to induce retinal apoptosis, while hinax 776(25pL) was administered intranasally to C57BL/6J mice. FIG. 1[ A-B ] shows a purely representative cSLO image of the retina at baseline (FIG. 1A) and a representative cSLO image of a DMSO-infested (FIG. 1B) retina prior to treatment. The same eyes were imaged 3 hours after treatment with 4% DMSO injection (figure ID) and intranasal Anx776 (figure 1C and figure ID). DARC spots (panel ID) were clearly visible in DMSO-treated eyes, providing first evidence that: intranasal administration of Anx776 may reach the retina at a concentration sufficient to label apoptotic retinal cells in a manner similar to intravenous or intravitreal administration of DARC. Manual quantification of DARC points indicated: increased eye apoptosis following intravitreal DMSO injury was consistent with previous observations of intravitreal injection of DARC in the model (approzike et al, 2005).

We hypothesize that the Anx776 inhalation cycle is followed by systemic absorption into the retina. Our experimental and clinical data with Anx776 demonstrated that systemic Anx776 could reach the retina after intravenous administration (coledilol et al, 2017).

Anx776 can be easily formulated as a lyophilized powder (with trehalose as cryoprotectant) that retains PS binding activity for up to 50 days after storage at 25 ℃ in the dark (fig IF). Anx776 powders containing up to 10mg/mL of Anx776 have been prepared, which can be rehydrated prior to IN delivery, or administered directly as a powder formulation.

b) The small fluorescent molecules currently used to diagnose retinal diseases enter the circulatory system rapidly enough to be used for diagnosis after intranasal application

It has been observed that the fluorescent protein Anx776(36kDa) enters the circulation following nasal administration, and we next attempted to determine whether small fluorescent molecules (376 Da and 775Da for sodium fluorescein and ICG, respectively) currently administered intravenously to diagnose retinal disease could also be delivered in this manner. Intranasal administration of either sodium fluorescein (fig. 2) or ICG (fig. 3) resulted in rapid accumulation of fluorescence in retinal blood vessels and choroid. The image obtained by cSLO is identical to the image obtained after a previous intravenous injection of the same fluorescent agent (kuma et al, 2014). Because of topical administration, fewer doses may be required and the onset of action is faster than in formulations where the agent is administered systemically.

Reference to the literature

M.F.Cordeiro,E.M.Normando,M.J.Cardoso,S.Miodragovic,S.Jeylani,B.M.Davis,L.Guo,S.Ourselin,R.A'Hern,P.A.Bloom,Brain 2017,274,61.

J.J.Jorzik,A.Bindewald,S.Dithmar,F.G.Holz,Retina 2005,25,405.

S.Kumar,Z.Berriochoa,A.D.Jones,Y.Fu,J.Vis.Exp.2014,e51061.

De Saint-Hubert M,Prinsen K,Mortelmans L,Verbruggen A,Mottaghy FM.Methods.2009 Jun；48(2):178-87

JF.Tait,C.Smith,FG.Blankenberg.J Nucl Med May 1,2005 vol.46no.5 807-815

Claims

1.A fluorescent marker for diagnosing retinal integrity in CNS diseases, wherein said fluorescent marker is delivered by intranasal administration.

2. The fluorescent marker for diagnosing retinal integrity for CNS diseases according to claim 1, wherein said fluorescent marker is in the form of a powder, suspension or solution.

3. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to claim 1 or 2, wherein said fluorescent marker is a marker of retinal vascular integrity.

4. The fluorescent marker for diagnosing retinal integrity for CNS diseases according to any one of claims 1 to 3, wherein said fluorescent marker has a molecular weight of about 2kDa or less.

5. A fluorescent marker for diagnosing retinal integrity for CNS disease according to any one of claims 1 to 4 wherein the fluorescent marker is selected from one or more of sodium fluorescein or indocyanine green (ICG).

6. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to claim 1 or 2, wherein said fluorescent marker is a marker of retinal cell integrity.

7. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to claim 1,2 or 6, wherein said fluorescent marker comprises a fluorescent tag and a marker of one or more of apoptosis, necrosis, cellular activity, cellular stress or protein aggregation.

8. The fluorescent marker for diagnosing retinal integrity for CNS diseases according to claim 6 or 7, wherein said fluorescent tag has an emission wavelength of about 400nm to about 1000 nm.

9. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to any one of claims 6 to 8, wherein said fluorescent tag is selected from one or more of sodium fluorescein, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488 and D-781.

10. A fluorescent marker for diagnosing retinal integrity in a CNS disease according to any one of claims 6 to 9, wherein said apoptotic marker is selected from one or more of annexin, the C2A domain of synaptophin I, doramectin, non-peptide based isatin sulfonamide analogues, such as WC-II-89 and ApoSense, such as NST-732, DDC and ML-10.

11. A fluorescent marker for diagnosing retinal integrity for a CNS disease according to claim 10, wherein said annexin is selected from one or more of annexin 2, annexin 5, annexin 6, annexin 11 or annexin 128.

12. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to any one of claims 6 to 9, wherein the necrosis marker is selected from the group consisting of Propidium Iodide (PI), pyrophosphate, antimyosin, oxalate, hypericin monocarboxylic acid, palmitic acid, bis-hydrazide-bis-DTPA palmitic acid, 99 mTc-pyrophosphate, 111-antimyosin, 99 mTc-gluconate and methylene blue.

13. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to any one of claims 6 to 9, wherein the marker of cellular activity is selected from one or more of membrane dyes, mitochondrial dyes, autophagy dyes, necrosis dyes and calcium flux.

14. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to any one of claims 6 to 9, wherein the marker of cellular stress is selected from one or more of lipid peroxidation, Glutathione (GSH) or Reactive Oxygen Species (ROS), such as superoxide, peroxyl radical, hydrogen peroxide, hydroxyl, peroxynitrite.

15. A fluorescent marker for diagnosing retinal integrity for CNS diseases according to any one of claims 6 to 9, wherein said marker of protein aggregation is selected from one or more of congo red, curcumin or thioflavin S.

16. A fluorescent marker for diagnosing retinal integrity in a CNS disease according to any one of claims 1 to 15 wherein the CNS disease is inflammatory (such as arthritis or granuloma), infectious (such as virus, encephalitis or bacteria), vascular (such as angiogenesis, occlusion or metabolism), or degenerative (such as glaucoma, age-related macular degeneration (AMD), alzheimer's disease, or parkinson's disease).

17. A method of diagnosing a CNS disease comprising administering to a subject a fluorescent marker of retinal integrity and generating an image of the eye of the subject, wherein the fluorescent marker is delivered by intranasal administration.

18. The method of claim 17, wherein the fluorescent marker is in the form of a powder, a suspension, or a solution.

19. The method of claim 17 or 18, wherein the fluorescent marker is a marker of retinal vascular integrity.

20. The method of any one of claims 17 to 19, wherein the fluorescent label has a molecular weight of about 2kDa or less.

21. The method of any one of claims 17 to 20, wherein the fluorescent label is selected from one or more of fluorescein sodium or indocyanine green (ICG).

22. The method of claim 17 or 18, wherein the fluorescent marker is a marker of retinal cell integrity.

23. The method of claim 17, 18 or 22, wherein the fluorescent label comprises a fluorescent tag and a label of one or more of apoptosis, necrosis, cellular activity, cellular stress or protein aggregation.

24. The method of claim 22 or 23, wherein the fluorescent tag has an emission wavelength of about 400nm to about 1000 nm.

25. The method of any one of claims 22-24, wherein the fluorescent tag is selected from one or more of fluorescein sodium, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488, and D-781.

26. The method of any one of claims 22 to 25, wherein the apoptosis marker is selected from one or more of annexin, the C2A domain of synaptophin I, doramectin, non-peptide based isatin sulfonamide analogs, such as WC-II-89 and ApoSense, such as NST-732, DDC and ML-10.

27. A method according to claim 26 wherein the annexin is selected from one or more of annexin 2, annexin 5, annexin 6, annexin 11 or annexin 128.

28. The method of any one of claims 22 to 25, wherein the necrosis marker is selected from one or more of Propidium Iodide (PI), pyrophosphate, antimyosin, oxalate, hypericin monocarboxylic acid, palmitic acid, dihydrazide-bis-DTPA palmitic acid, 99 mTc-pyrophosphate, 111-antimyosin, 99 mTc-gluconate, and methylene blue.

29. The method of any one of claims 22 to 25, wherein the marker of cellular activity is selected from one or more of a membrane stain, a mitochondrial stain, an autophagy stain, a necrosis stain and calcium flux.

30. The method of any one of claims 22 to 25, wherein the marker of cellular stress is selected from one or more of lipid peroxidation, Glutathione (GSH) or Reactive Oxygen Species (ROS), such as superoxide, peroxyl radical, hydrogen peroxide, hydroxyl, peroxynitrite.

31. The method of any one of claims 22 to 25, wherein the marker of protein aggregation is selected from one or more of congo red, curcumin or thioflavin S.

32. A method according to any one of claims 17 to 31 wherein said CNS disorder is inflammatory (such as arthritis or granuloma), infectious (such as virus, encephalitis or bacteria), vascular (such as angiogenesis, occlusion or metabolism), or degenerative (such as glaucoma, age-related macular degeneration (AMD), alzheimer's disease or parkinson's disease).

33. A pharmaceutical composition comprising annexin or a functional fragment or derivative thereof bound to a compound of 2kDa or less, wherein the pharmaceutical composition comprises annexin or a functional fragment or derivative thereof bound at a concentration of at least 5 mg/ml.

34. The pharmaceutical composition of claim 33, wherein the annexin or the functional fragment or derivative thereof is annexin 128 and the compound of 2kDa or less is Dy 776.